UNIVERSITI TEKNOLOGI MARA
DETERMINATION OF MAJOR FRAGRANCE COMPOUNDS AND DEVELOPMENT OF CELL
CULTURES OF Citrus grandis (OSBECK.) FLOWERS
SUHANA BT ZAKARIA
MSc
October 2009
Candidate's Declaration
I declarethat the work in thisthesiswas carried outin accordance with the regulations of Universiti Teknologi MARA.Itis originaland is the result of my own work,unless otherwiseindicated or acknowledged as referenced work. Thisthesis has not been submitted to anyother academic institution or non-academicinstitution for any other degree or qualification.
In theeventthat my thesisbe found to violate the conditions mentioned above,I voluntarily waive the right ofconferment of mydegree and agree to be subjected to the discipl inaryrulesand regulationsofUniversitiTeknologi MARA.
Nameof Candidate Candidate'sID No.
Programm e Faculty Thesis Title
Signatureof Candidate Date
: Suhana bt Zakaria :20051068 10
:Master of Science (Biology) :Facultyof Applied Sciences
:Determinationof Major FragranceCompounds and Development of Cell Cultures of Citrus grandis (Osbeck.)Flowers
ABSTRACT
Citrus grandis flowers possesses a strong floral-, jasmine-and orange-like aroma that has a potential in perfumery industries. Three extraction techniques that is hydrodistillation,Soxhlet extraction and solid phase micro extraction (SPME) were coupling with gas chromatograph-massspectrometry (GC-MS) for analysis of fragrance compounds from this flowers.About 120 compounds were detected in bud and blossom of these flowers, of which the five major fragrance compounds were~-myrcene, limonene, ocimene, linalool and caryophyllene. In hydrodistillation, ocimene and linalool detected in blossom were higher than in bud with 4.57%. However,Soxhlet extraction showed that bud contained high percent of limonene (27.25%),ocimene (5.55%)and linalool (2.97%) compared to blossom. Three different SPME fibers were used in this study,namely,65 urn CAR/POMS, 75 urn PDMS/DVB and 100 urn POMS.
The best result was obtained by 100 urn POMS, that were then further analyzed for optimum sampling time.Itshowed 60 min fiber exposition time was the optimum sampling time to extract the major fragrance compounds from the headspace.In cell culture development,callus was successfully induced from different part ofC.grandis flowers like petal,sepal,style,ovary, pistil and cup base on Murashige and Skoog (MS) medium supplemented with sucrose (30gil)and various concentrations of hormones.It was found that different parts of flowers required different level of hormone for callus induction. The highest formation of callus were obtained from petal and sepal cultured on MS media supplemented with 1.5 mg/l kinetin. For the style and pistil, 50% of the explants had developed callus when cultured on MS media added with 0.05mg/l and 0.10 mg/l BAP respectively.On the other hand, sucrose agar media alone managed to induce callus formation from the cup base and ovary with success rate between 11± 1.29 to 25±4.19%.Callus obtained were then subjected to SPME for determination of compounds in the callus.Seven compounds were detected in the callus which some of the compounds shows the same compounds as detected in the fresh flowers.
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TABLE OF CONTENT
Page
ABSTRACT iii
ACKNOWLEDGEMENTS iv
TABLE OF CONTENTS LIST OF TABLES
LIST OF FIGURES xi
LIST OF PLATES xii
LIST OF APPENDICES xiii
GLOSSARY xiv
CHAPTER 1 : INTRODUCTION 1.1 Fragrance CompoundsCompositions 1.2 Citrusgrandis
1.3 Extraction Techniques 2
1.4 Plant TissueCultures 3
1.5 Obj ectives 4
CHAPTER 2 : LITERATURE REVIEW 2.1 Fragrance Compounds Compositions 2.2 Fragrance Compounds fromCitrus
2.3 The Taxanomy and Origin ofCitrus grandis(L.)Osbeck
2.4 Pomelo Plantation in Malaysia 8
2.5 Extraction Techniques for the Analysis of Fragrance Compounds 10
2.5. 1 Hydrodistilla tion 11
2.5.2 Soxhlet Extraction 12
2.5.3 Solid Phase Micro Extraction(SPME) 13
2.6 Plant Tissue Culture 17
2.7 CallusCulture 18
CHAPTERl
INTRODUCTI ON
1.1 Fragrance Compounds Compositions
Essential oilsare the highly concentrated,volatile oils of plant materials that have strong aromatic components. They are made up of different fragrance compounds that occur naturally in the plant and can be found in anypart of plants like in leaves, flowers,roots, seeds,fruit pulpsor peels (Lawless,2002).In flowers of many plant species,the complex mixturesof volatile compounds can be found in various floral organs such as in stigma,style,ovary,petal andsepa l. The combination of the constituentsof thisscent mixtur egives each flowering plant speciesa unique fragrance.An example,C. grandis flower wasdescribedas havingafloral-,jasmine-and orange-like fragrance (Svoboda&
Greenaway, 2003 ). Citrus specieshas obtainedgreat interest among researcher on their valuable compounds especially for flavonoid and monoterpenoid production.The monoterpenoidscompoundsare themajor component of many essential oils and as such, have economic importance asflavours and perfumes (Svoboda&Greenaway,2003).
Nowadays,mostsecondary metabolites are obtained directly from the plant tissue whose availability maybe limited (Ibrahim, Hussein,Hamzah,Peng,&Simoh,2005). Through out the world,there are over4000 of plant species and more than 750,000tonnes annually are being collected from the wild (Svoboda&Greenaway,2003).This plant material is a .sourceof natural products used in various ways including as fragrance. However,there is a limitation to depend on plant materials for continuous supply.Due to theincreasing demand of fragrance compounds, the dependence on plant material for continuoussupply was limited.IndustryMarket Research Online reported that the
